Browsing by Author "Shi, Yan"

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    Contact-dependent delivery of IL-2 by dendritic cells to CD4 T cells in the contraction phase promotes their long-term survival
    (2019-11-05) Tong, Dan; Zhang, Li; Ning, Fei; Xu, Ying; Hu, Xiaoyu; Shi, Yan
    Abstract Common γ chain cytokines are important for immune memory formation. Among them, the role of IL-2 remains to be fully explored. It has been suggested that this cytokine is critically needed in the late phase of primary CD4 T cell activation. Lack of IL-2 at this stage sets for a diminished recall response in subsequent challenges. However, as IL-2 peak production is over at this point, the source and the exact mechanism that promotes its production remain elusive. We report here that resting, previously antigen-stimulated CD4 T cells maintain a minimalist response to dendritic cells after their peak activation in vitro. This subtle activation event may be induced by DCs without overt presence of antigen and appears to be stronger if IL-2 comes from the same dendritic cells. This encounter reactivates a miniature IL-2 production and leads a gene expression profile change in these previously activated CD4 T cells. The CD4 T cells so experienced show enhanced reactivation intensity upon secondary challenges later on. Although mostly relying on in vitro evidence, our work may implicate a subtle programing for CD4 T cell survival after primary activation in vivo.
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    Efficient Clearance of Inhaled Nanoparticles Depends on Strong Adhesion to the Epithelium: The Role of Hydrophilicity, Coating, Size, and Shape
    (2023-09-14) Bogari, Nawaf Nasir; Amrein, Matthias; Green, Francis; Yates, Robin; Shi, Yan; Schriemer, David; Hubbs, Ann
    Health alerts regarding high levels of fine particles in ambient air are increasingly common, reflecting a significant worldwide crisis. These particles contribute substantially to premature death, a problem only expected to grow. Among these, nanoparticles pose a particular threat, linked to respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and cancer as well as extrapulmonary effects. The health implications of nanoparticles differ, rendering simple particle concentration nearly meaningless. In this thesis, we focused on understanding the accumulation of nanoparticles in the lung, which is a critical aspect of their toxicity. We developed in-vitro assays to predict whether particles remain in the alveolar lumen to be cleared through alveolar and airway pathways, and which particles traverse the alveolar epithelium to induce interstitial lung disease or enter the blood and lymphatic vessels to cause systemic effects. We hypothesized that the fate of inhaled nanoparticles in the alveolar lung primarily depends on their adhesion strength to alveolar epithelium and the physical/chemical characteristics of the particle. To test this, we employed atomic force microscopy (AFM) for adhesion force measurement, transmission electron microscopy (TEM) to correlate adhesion strength to nanoparticle uptake, and confocal laser scanning microscopy (CLSM) to study nanoparticle translocation. Characteristics of nanoparticles, including protein corona coating, alveolar surfactant coating, hydrophobicity/hydrophilicity, size and geometry were studied in the presence or absence of pharmacological blockers to determine endocytic mechanism(s) involved in the interaction with the epithelium. Results showed that the adhesion of nanoparticles to the epithelial cells was an active process, and the strength of adhesion to the epithelium correlated directly to their uptake and transcytosis. Amorphous silica nanoparticles (ASN) with 15 nm in diameter were found to adhere strongly and translocate across the epithelium, whereas nanocarbon black particles 15 nm in diameter (nCB15) exhibited weak adhesion and remained in the alveolar lumen. Interestingly, commonly studied zeta potential had no influence on the interaction, whereas particle coating with surfactant increased their potential to accumulate in the alveolar lumen. Rendering ASN particles hydrophobic reduced their adhesion to the epithelium. The size of nanoparticles was linked to how cells perceived nanoparticles, with particles larger than 150 nm being endocytosed in a clathrin-enhanced mechanism, while particles less than 150 nm were taken up in a caveolin-enhanced mechanism. The epithelium did not show a preferred endocytic mechanism with respect to clathrin or caveolin for silica nanorods (SNR) yet showed a cytotoxic response to these elongated particles. This work contributes to the development of an effective framework for assessing the potential risks of inhaled nanoparticles, and our novel approach of categorization can support public health policies aiming to reduce exposure to nanoparticles in various environments.
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    Force Based Comparison Between Naïve and Activated CD4+ T Cells during Interaction with Dendritic Cells
    (2014-04-11) ALGHAMDI, ENAAM; Shi, Yan
    Antigen recognition requires the communication between T cells and antigen presenting cells (APC) such as dendritic cells (DC). Although there is progress in understanding molecular components at the intercellular contact, the specific role of the physical cross-junctional adhesion between CD4+ T cells and DCs is not fully understood. In this study, we investigate cell-to-cell contact mediated by adhesion molecules between these cells in order to elicit the mechanism involved. Lymphocyte function-associated antigen-1 (LFA-1) adhesion molecule on the surface of T cells interacts with intracellular adhesion molecule-1 (ICAM-1) on the APC. This adhesion pair has been shown to be essential for the contact between the cells. The role of this adhesion pair and its expression and regulation on naïve CD4+ T cells has not been compared to its role on activated T cells. Using atomic force microscopy (AFM) we investigated the increase in interaction forces between naïve CD4+ T cells and DCs. These forces were compared to those involving activated T cells to see the difference between naïve and activated T cells in their adhesion properties with DCs, which involves LFA-1 integrin molecule. Understanding the role of LFA-1 interaction with ICAM-1 provides further insight into therapies using CD4+ T cells.
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    Identification and Characterization of Moesin-, PIP2-mediated Solid Particle Phagocytosis
    (2018-08-09) Tu, Zhongyuan; Shi, Yan; Yates, Robin Michael; Amrein, Matthias; Prenner, Elmar J.; Botelho, Roberto
    Phagocytosis is the defining feature of professional phagocytes of the innate immune system. This function is typically carried out by phagocytic receptors on the cell surface. These receptors can mediate binding and engulfment of solid particles. However, these phagocytic receptors have evolved very recently in history comparing to phagocytosis as a conserved cellular function. This suggests a primordial form of phagocytosis might exist. Years ago, our laboratory uncovered an expected phagocytic mechanism that solid particle can bind to membrane lipids on phagocytes to trigger lipid sorting. Consequently, this can lead to phagocytosis akin to FcγR-based phagocytosis regarding its dependence on Immunoreceptor Tyrosine-based Activation Motif (ITAM), Src-family kinases, Syk, and phosphoinositide 3-kinase (PI3K). Based on these findings, we proposed a hypothetical mechanism for solid particle phagocytosis termed “Signaling Equivalent Platform” (SEP). In short, membrane engagement with solid structures, either via ligand/receptor binding or merely being stabilized by an approaching solid surface will lead to a shared downstream pathway with the same dependence on ITAM and Syk. Both modes of phagocytosis are equivalent for its activation by solid structures. However, the identity of the ITAM-containing molecule and the exact involvement of lipid during solid particle phagocytosis under SEP is still unclear. This thesis serves to strengthen the idea of SEP by identifying the ITAM-containing molecule and further characterizing the involvement of the ITAM-containing molecule and lipids during solid particle phagocytosis. We used a generic ITAM sequence as a probe and identified moesin as the ITAM-containing molecule from the mouse genome. We further demonstrated that a solid structure binding to the cell surface leads to autonomous accumulation of phosphatidylinositol 4, 5-bisphosphate (PIP2) to the site of contact, which attracts moesin, a conserved structural linker, to the plasma membrane. Moreover, Moesin, via its ITAM, is sufficient to activate phagocytic programming including Syk and downstream signaling that is virtually identical to that initiated by Fcγ receptors. Bioinformatic analysis suggested that this moesin-mediated signaling predates modern Fcγ and immune receptors. This thesis, therefore, reveals an evolutionarily conserved moesin-, PIP2-mediated signaling platform for the evolutionarily conserved phagocytosis that provides essential components for modern ITAM-based signaling cascades.
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    Observations in Lipid Membrane Systems
    (2015-09-18) Munro, Fay; Amrein, Matthias; Shi, Yan
    The plasma membrane is phase separated into a fluid (Ld) phase and a more ordered (Lo) phase. The latter exists as small “rafts” of specific lipid composition containing a host of signaling proteins. Lipid raft theory links the aggregation of rafts to a signaling event. In its current iteration, the theory is incomplete, as it does not explain how rafts form and how they aggregate and disperse again. These problems are addressed when the membrane is viewed as a critical system. Previous work using giant plasma membrane vesicles (GPMVs) used this framework to explain the dynamics of the rafts. We intended to show critical behavior as a factor for cell signaling. Critical behavior was not easily ascertained and further observations were made. We found a heterogeneous population with respect to the behavior of the vesicles. We categorized these observations with respect to the appearance of the lipid membrane to characterize the phenomenon.
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    Regulation of the NLRP3 Inflammasome by Cathepsin Z
    (2020-07-06) Campden, Rhiannon I; Yates, Robin M.; Hollenberg, Morley; Shi, Yan; Khrisendath, Chadee; Akkari, Leila
    The focus of this dissertation is on understanding how cathepsin Z is involved in the initiation of inflammation, and specifically, how cathepsin Z contributes to the production of interleukin (IL)-1? following NLRP3 inflammasome activation. IL-1? is a pro-inflammatory cytokine involved in the recruitment of immune cells and the polarization of TH17 cells. Activation of the NLRP3 inflammasome, which is a macromolecular complex composed of a receptor (NLRP3), an adaptor protein (ASC), and the protease caspase-1, leads to cleavage and release of IL-1?. Cathepsin Z contains dual functions as a C-terminal exopeptidase and an integrin-binding protein. Initially, we explored the involvement of cathepsin Z in the development of inflammation in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). We found that cathepsin Z is required for the recruitment of immune cells into the central nervous system (CNS). Here, we found cathepsin Z is involved in TH17 polarization due to reduced IL-1?, but not antigen processing, T cell activation nor T cell migration. This led us to investigate the mechanism through which cathepsin Z regulates IL-1? production. We found that extracellular cathepsin Z signals through the ?5 integrin following NLRP3 inflammasome activation. Furthermore, the function of cathepsin Z in this context was dependent on the integrin-binding domain of cathepsin Z and not its proteolytic activity. This reveals another mechanism of NLRP3 regulation through an endogenous extracellular protein that signals through integrins. Finally, we employed proteomic and degradomic approaches following NLRP3 inflammasome activation in the absence of NLRP3 to gain a deeper understanding of the proteins involved in NLRP3 inflammasome activation. In the absence of NLRP3, caspase-1 and IL-1? are degraded, while caspase-4 is stabilized. Further, ROS levels are lower following NLRP3 inflammasome activation with silica in the absence of NLRP3, and the expression of several mitochondrial proteins are deregulated, suggesting the importance of the mitochondria and ROS signalling in NLRP3 inflammasome activation. Collectively, this dissertation reveals a novel extracellular regulatory pathway of the NLRP3 inflammasome and further outlines the signalling function of cathepsin Z in inflammation. This dissertation also presents a dataset of proteins for investigation in NLRP3 inflammasome activation.
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    The nature of regulatory t cell interactions with dendritic cells
    (2012) Mucsi, Ashley Dawn; Shi, Yan
    The immune system has developed several mechanisms responsible for discriminating against self and non-self antigens and preventing autoimmune responses from occurring within the body. Inactivation of auto-reactive lymphocytes and elimination of these cells prevents the generation of autoimmune responses ensuring that tolerance results in the periphery. A specialized subpopulation of CD4+ T cells, regulatory T cells (Tregs ), maintains peripheral self-tolerance through a diverse set of mechanisms. The mechanisms utilized by regulatory T cells are vast; however, the mechanisms by which Tregs modulate their suppressive function are not yet fully understood. Utilizing atomic force microscopy, we demonstrate that IL-2 stimulated Tregs interact intensely with dendritic cells (DC), which is dependent upon the LFA-1/ICAM-1 adhesion molecule. We propose that LF A-1 regulation differs between Tregs and CD4+ CD25- T cells, allowing Tregs to interact intensely with DCs abrogating any interaction with antigen­specific T cells. Our results illustrate how Tregs interact with DCs and its association with LF A-1 avidity regulation.
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    Toll-Like Receptors: Role in Dermatological Disease
    (2010-08-22) Hari, Aswin; Flach, Tracy L.; Shi, Yan; Mydlarski, P. Régine
    Toll-like receptors (TLRs) are a class of conserved receptors that recognize pathogen-associated molecular patterns (PAMPs) present in microbes. In humans, at least ten TLRs have been identified, and their recognition targets range from bacterial endotoxins to lipopeptides, DNA, dsRNA, ssRNA, fungal products, and several host factors. Of dermatological interest, these receptors are expressed on several skin cells including keratinocytes, melanocytes, and Langerhans cells. TLRs are essential in identifying microbial products and are known to link the innate and adaptive immune systems. Over the years, there have been significant advances in our understanding of TLRs in skin inflammation, cutaneous malignancies, and defence mechanisms. In this paper, we will describe the association between TLRs and various skin pathologies and discuss proposed TLR therapeutics.
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    Toll-Like Receptors: Role in Dermatological Disease
    (Hindawi Publishing Corporation, 2010-07-01) Hari, Aswin; Flach, Tracy L.; Shi, Yan; Mydlarski, P. Régine
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    Unraveling mechanisms behind cross presentation during phagocytic signaling
    (2014-01-29) Hari, Aswin; Shi, Yan
    MHC class I antigens come from endogenous syntheses, while class II antigens are obtained extracellularly via endocytosis. Cross presentation is the link between the two by which external antigens are targeted to the MHC class I pathway. It is generally accepted that particulate antigens are more efficient in this pathway switch. However the reason behind this phenomenon is unknown. We report that dendritic cell engagement of a phagocytic target limits endocytic maturation and inhibits the associated proteolytic activities. In this scenario, early endosomes show reduced progression towards late endosomes/lysosomes and remain spatially close to the cell membrane. In phagocytosis, the microtubular (MT) system, including tubulin filaments and microtubule organization centers (MTOCs), are heavily skewed toward the engulfed particulate matter; the remaining cytoplasmic volumes are relatively devoid of MT presence. This is accompanied by a reduced centripetal movement inward and the maturation of endosomes. The antigen processing in these arrested endosomes is under the control of Nicotinamide adenine dinucleotide phosphate-oxidase (NAPDH)-associated ROS. We also show that cathepsin S is responsible for the generation of the class I epitope for cross presentation. The rerouted antigen presentation is at least 40 fold more efficient than the same amount of antigen delivered through the phagocytic pathway, and is operational in vivo. Our results suggest that in DCs in addition to solid structure uptake, phagocytosis directs a coordinated set of enzymatic and cytoskeletal events that regulate endosomal trafficking and maturation. As a consequence, external soluble antigens are driven away from their conventional MHC class II processing, into the class I cross presentation pathway.